This project is designed to investigate important questions about the mechanism and regulation of transcription initiation in Escherichia coli. The experiments are designed to provide a fundamental biochemical understanding of several complex phenomena involved in the recognition of E. coli promoters by RNA polymerase. A series of studies will characterize the "closed complex", an important reaction intermediate in the initiation process. Experiments performed in vitro will also characterize the properties of a mutant sigma subunit that displays altered promoter recognition in vivo. The effects of DNA supercoiling on promoter recognition will be explored using a newly-developed assay for topoisomer selectivity. This novel approach will be extended in a study of positive regulation by cap*cAMP at the lactose and galactose operon promoters. The modulation of promoter selectivity by the galactose repressor at the gal promoters will also be characterized. The structural determinants of RNA-RNA pairing will also be determined in an example of antisense RNA gene regulation. In this case the regulation occurs primarily at the translation initiation steps, however, the principles for RNA-RNA pairing that emerge will be applicable to many other steps in gene expression and its regulation. The answers to the questions we have posed will contribute to the biochemical description of the control of gene expression.